非揮發性磷酸緩衝溶液於核苷酸之毛細電泳質譜研究

Abstract

核苷酸為生物體內重要生物分子之一，結構為鹼基、一個五碳醣和一至三個磷酸根。文獻中指出以毛細管電泳分離此類化合物時，核苷酸上的磷酸根會和熔融玻璃毛細管上之矽氧基產生作用力造成吸附而造成分析峰變形。當以磷酸鹽類作為緩衝溶液時，可以有效改善此吸附問題得到較對稱的峰形。但非揮發性磷酸鹽溶液在電灑法質譜上，會造成嚴重的化學訊號干擾，及抑制分析物的訊號。為了保有磷酸鹽類在分離上的好處且能銜接毛細電泳質譜，本研究將使用先前已開發的低流速鞘流溶液介面，及低流速鞘流—液體接合介面(liquid-junction /low-flow interface)於毛細電泳質譜上。使用添加30%異丙醇的低濃度磷酸鹽緩衝溶液，以低流速鞘流介面銜接毛細電泳質譜分析核苷酸，可成功分析三種核苷酸，且磷酸根會持續退回進樣槽而減少抑制，但為了保有降低核苷酸吸附的效用，每次分析前皆需重新補充磷酸根，且分析時間較長。進一步使用低流速鞘流－液體接合介面銜接毛細電泳質譜分析核苷酸，可於分析過程中持續由進樣端補充磷酸根，且可在不影響最佳分離條件下，於液體接合槽與銜接管中添加適當比例的異丙醇，提供穩定的電噴灑及控制銜接管中電滲流的大小以擋下磷酸根，不僅可改善訊號抑制及干擾的問題，且保留磷酸鹽溶液於電泳分離上之好處。

Nucleotides, composed of a nucleobase, a five-carbon sugar, and one to three phosphate groups, are one kind of very important biomolecules in the living organisms. When analyzing these compounds with capillary electrophoresis (CE), the silanol groups on the capillary wall interact with phosphate groups, thus causing distortion of electrophoretic peaks. Phosphate running buffers can effectively reduce the adsorptive interaction and provide better resolution and peak shape. However, the nonvolatile phosphate buffer would cause severe signal suppressions and chemical interferences in electrospray ionization mass spectrometry spectrum (ESI-MS). In this study, both the low-flow interface and the liquid-junction/low-flow interface were utilized to solve this problem. By using lower concentration phosphate buffer with the addition of isopropanol in CE-MS, we have successfully analyzed nucleotides using a low-flow interface. Under the condition, phosphate ions would migrate to the inlet resulting in less signal suppression. However, due to the addition of isopropanol, the supplement of phosphate ions is needed, and the analysis time also becomes much longer. Liquid-junction/low-flow interface was used to alleviate the problem of long analysis time and re-codition the column. By controlling the electroosmotic flow (EOF) of the connecting column with the addition of isopropanol, phosphate ions would stay in the liquid junction reservoir, whereas the nucleotides would migrate toward the ion source. This approach not only retains the short separation time but also alleviates the signal suppression and interference in MS spectra.